Lung adenocarcinoma, one of the most prevalent malignancies, underscores the urgency of identifying genes linked to its proliferation and prognosis for developing targeted therapies. In this study, we performed genome-wide CRISPR/Cas9 screening both in vitro and in vivo, and subsequently cross-referenced the findings with prognosis-related genes in lung adenocarcinoma. The screening results revealed that DEFB1 promotes lung adenocarcinoma proliferation, while our integrated analysis of single-cell sequencing, multiplex immunohistochemistry, TCGA, and GEO data concurrently demonstrated elevated DEFB1 expression in cancer cells, along with a negative correlation between DEFB1 expression and patient survival. Subsequently, functional studies employing DEFB1 knockout cells, re-expressed DEFB1 cells, and knockout cells supplemented with exogenous DEFB1 revealed that DEFB1 significantly enhances cell proliferation, migration, and invasion. Co-immunoprecipitation combined with mass spectrometry experiments was performed to uncover the mechanism of DEFB1, demonstrating that it interacts with Periplakin (PPL) to induce epithelial-to-mesenchymal transition (EMT) and proliferation, while simultaneously binding to Macrophage Migration Inhibitory Factor (MIF) to enhance M2 macrophage polarization. Furthermore, we developed multiple anti-DEFB1 monoclonal antibodies and found one of them, mAb-5, potently blocked DEFB1's function and inhibited lung adenocarcinoma progression in cell lines, organoids, xenografts, and spontaneous lung cancer models, while maintaining a favorable safety profile. Overall, our study identifies DEFB1 as a novel driver of lung adenocarcinoma, and the anti-DEFB1 monoclonal antibody mAb-5 emerges as a promising therapeutic candidate with significant potential.